Surface binding of galectin family members has the potential to link distinct glycan structures to growth regulation. Therefore, we addressed the antiproliferative potential of galectin-1 (Gal-1) in a panel of carcinoma cell lines. We discovered growth inhibition by Gal-1 in epithelial tumor cell lines from different origins and provide evidence that this effect requires functional interaction with the α5β1 integrin. Antiproliferative effects result from inhibition of the Ras-MEK-ERK pathway and consecutive transcriptional induction of p27. We have further identified two Sp1-binding sites in the p27 promoter as crucial for Gal-1 responsiveness. Inhibition of the Ras-MEK-ERK cascade by Gal-1 increased Sp1 transactivation and DNA binding due to reduced threonine phosphorylation of Sp1. Furthermore, Gal-1 induced p21 transcription and selectively increased p27 protein stability. Gal-1-mediated accumulation of p27 and p21 inhibited cyclin-dependent kinase 2 activity and ultimately resulted in G₁ cell cycle arrest and growth inhibition. These data define a novel mechanism whereby Gal-1 regulates epithelial tumor cell homeostasis via carbohydrate-dependent interaction with the α5β1 integrin.

The tumor suppressor p16^INK4ahas functions beyond cell‐cycle control via cyclin‐dependent kinases. A coordinated remodeling ofN‐ andO‐glycosylation, and an increase in the presentation of the endogenous lectin galectin‐1 sensing these changes on the surface of p16^INK4a‐expressing pancreatic carcinoma cells (Capan‐1), lead to potent pro‐anoikis signals. We show that the p16^INK4a‐dependent impact on growth‐regulatory lectins is not limited to galectin‐1, but also concerns galectin‐3. By monitoring its expression in relation to p16^INK4astatus, as well as running anoikis assays with galectin‐3 and cell transfectants with up‐ or downregulated lectin expression, a negative correlation between anoikis and the presence of this lectin was established. Nuclear run‐off and northern blotting experiments revealed an effect of the presence of p16^INK4aon steady‐state levels of galectin‐3‐specific mRNA that differed from decreasing the transcriptional rate. On the cell surface, galectin‐3 interferes with galectin‐1, which initiates signaling toward its pro‐anoikis activity via caspase‐8 activation. The detected opposite effects of p16^INK4aat the levels of growth‐regulatory galectins‐1 and ‐3 shift the status markedly towards the galectin‐1‐dependent pro‐anoikis activity. A previously undescribed orchestrated fine‐tuning of this effector system by a tumor suppressor is discovered.

Purpose: Inhibition of angiogenesis represents a promising therapeutic strategy in neuroendocrine tumors. Angiopoietin-2 (Ang-2), a ligand of the endothelial tyrosine kinase Tie-2, is emerging as a key regulator of vascular remodeling during tumorangiogenesis. We therefore addressed the expression and biological significance of Ang-2 in human neuroendocrine tumors. Experimental Design: Surgical specimens and serum from neuroendocrine tumor patients were used to determine Ang-2 expression by in situ hybridization or ELISA (circulating Ang-2). Ang-2 biological effects were evaluated following stable transfection into BON human pancreatic neuroendocrine tumor cells. BON clones were grown as orthotopic xenografts in nude mice to determine tumor growth and abdominal metastatic spread. Further analyses included microvessel density, lymphatic vessel density, and nodal invasion. Results: Specimens from pancreatic neuroendocrine tumors and nontransformed pancreatic tissue revealed uniform expression of Ang-2 mRNA in endothelial cells. In contrast, epithelial expression of Ang-2 mRNA occurred exclusively in neuroendocrine tumors. Overexpression of Ang-2 in BON orthotopic xenografts did not affect primary tumor growth, although successful Ang-2 induction was confirmed from elevated serum levels. However, increased microvessel density and enhanced lymphatic metastasis were evident in Ang-2–expressing tumors, indicating a functional role of Ang-2 in experimental neuroendocrine tumors. Consistent with this notion, circulating Ang-2 was significantly elevated in neuroendocrine tumor patients compared with healthy controls. Circulating Ang-2 furthermore correlated with metastatic versus localized disease. The highest Ang-2 concentrations occurred in patients with liver metastasis, and concentrations ≥75th percentile predicted shorter survival (P = 0.0003). Conclusion: Induction of Ang-2 in neuroendocrine tumors represents a clinically relevant pathomechanism of disease progression and constitutes an adverse prognostic marker. Clin Cancer Res; 16(2); 420–9.

Anoikis resistance is a hallmark of transformed epithelial cells. Here, we show that treatment of anoikis-resistant carcinoma cell lines with the endogenous lectin galectin-1 (Gal-1) promoted apoptosis via interaction with the unligated fibronectin receptor α₅β₁-integrin. Gal-1 efficiency correlated with expression of α₅β₁-integrin, and transfection of the α₅-subunit into deficient cell lines conferred Gal-1 binding and anoikis stimulation. Furthermore, Gal-1 and the α₅- and β₁-integrin subunits co-precipitated in Gal-1-stimulated cells undergoing anoikis. Other members of the galectin family failed to be active. The functional interaction between Gal-1 and α₅β₁-integrin was glycan dependent with α2,6-sialylation representing a switch-off signal. Desialylation of cell surface glycans resulted in increased electrophoretic mobility of α₅β₁-integrin and facilitated Gal-1 binding and anoikis stimulation. On the level of signaling, Gal-1-stimulated anoikis was prevented by filipin, which impaired the internalization of α₅β₁-integrin via cholesterol-enriched microdomains, and by pretreatment with a caspase-8 inhibitor. We propose that Gal-1/α₅β₁-integrin interaction participates in the control of epithelial integrity and integrin sialylation may enable carcinoma cells to evade this Gal-1-dependent control mechanism.

Lymphatic metastasis constitutes a critical route of disease dissemination, which limits the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). As lymphangiogenesis has been implicated in stimulation of lymphatic metastasis by vascular endothelial growth factor‐C (VEGF‐C) and VEGF‐D, we studied the effect of the angioregulatory growth factor angiopoietin‐2 (Ang‐2) on PDAC progression. Ang‐2 was found to be expressed in transformed cells of human PDAC specimens, with corresponding Tie‐2 receptors present on blood and lymphatic endothelium. In vitro in PDAC cells, Ang‐2 was subject to autocrine/paracrine TGF‐β stimulation (2‐fold induction, P = 0.0106) acting on the –61‐ to +476‐bp element of the human Ang‐2 promoter. In turn, Ang‐2 regulated the expression of genes involved in cell motility and tumor suppression. Orthotopic PDAC xenografts with forced expression of Ang‐2, but not Ang‐1, displayed increased blood and lymphatic vessel density, and an enhanced rate of lymphatic metastasis (6.7‐ to 9.1‐fold, P <0.01), which was prevented by sequestration of Ang‐2 via coexpression of soluble Tie‐2. Notably, elevated circulating Ang‐2 in patients with PDAC correlated with the extent of lymphatic metastasis. Furthermore, median survival was reduced from 28.4 to 7.7 mo in patients with circulating Ang‐2 ≥ 75th percentile ( P =0.0005). These findings indicate that Ang‐2 participates in the control of lymphatic metastasis, constitutes a noninvasive prognostic biomarker, and may provide an accessible therapeutic target in PDAC.—Schulz, P., Fischer, C., Detjen, K. M., Rieke, S., Hilfenhaus, G., von Marschall, Z., Böhmig, M., Koch, I., Kehrberger, J., Hauff, P., Thierauch, K.‐H., Alves, F., Wiedenmann, B., Scholz, A. Angiopoietin‐2 drives lymphatic metastasis of pancreatic cancer. FASEB J. 25, 3325–3335 (2011). www.fasebj.org

Therapeutic options to inhibit growth of human NETs of the GEP system are limited. Since NSAIDs might provide an antiproliferative treatment alternative with acceptable toxicity, we examined the effects of different NSAIDs on growth and survival in a representative set of human GEP NET cell lines. Growth and apoptosis were determined based on cell numbers, cell-cycle analyses, kinase assays, DNA fragmentation and PARP cleavage. Expression of COX and cell cycle–regulatory molecules was examined by immunoblotting and reporter gene assays. Depending on the drug and cell line investigated, NSAID treatment resulted in profound growth inhibition of GEP NET cells. Growth-inhibitory effects were achieved with either COX-2 selective (NS398) or unselective (indomethacin, sulindac) compounds. Cell-cycle analyses documented a G₁ arrest in NSAID-treated GEP NET populations. In addition, 100 μM sulindac or indomethacin induced apoptosis. All 3 COX inhibitors prevented CDK-2 activation. In parallel to the NSAID-mediated reduction of CDK-2 activity, p21^cip-1 promoter activity and cellular p21^cip-1 levels increased and p21^cip-1 was sequestered into CDK-2 complexes. Thus, the G₁ arrest likely resulted from p21^cip-1-dependent inhibition of CDK-2 activity. At therapeutically relevant concentrations, sulindac significantly reduced GEP NET cell numbers, whereas IFN-α and octreotide remained ineffective. The extent of growth inhibition in GEP NETs was comparable to the antiproliferative effects of sulindac in established NSAID-sensitive cell models. NSAIDs acted as potent antiproliferative agents in GEP NET cells via G₁ cell-cycle arrest and might therefore offer a therapeutic alternative to current treatment modalities.